This laboratory is involved in research in understanding the mechanisms whereby human tumor cells develop resistance to cytotoxic agents. In particular, we are concerned with identifying the genetic mechanisms involved in the development of drug resistance. We have isolated human breast cancer and human small cell lung cancer cells which are resistant to a number of cytotoxic agents including methotrexate, PALA, azauridine, pyrazofuran, and cadmium. Characterization of these resistant cells have revealed that, at least, in the first two instances (MTX and PALA) drug resistance in human tumor cells isolated in vitro is frequently associated with over-production of the target enzyme (DHFR and ATCase respectively), increased levels of messenger RNA coding for the target enzyme, and increased copies of the genes which code for these proteins. In the MTX breast cancer cells the amplified DHFR genes have apparently undergone a uniform gene rearrangement in the 5' (upstream) flanking sequences during the course of gene amplification. Ongoing studies include: 1) cloning and structural analysis of the amplified DHFR and comparison with the parental DHFR gene; 2) studies on the mechanisms of regulation of DHFR levels by estrogen and by MTX; 3) studies on the factors which affect the frequency of drug resistant in tumor cells; 4) analyses of the mechanisms of resistance to MTX in human tumor cells obtained directly from patients; 5) the transfer of drug resistant genes to other human cells; 6) analysis of the mechanisms of cross resistance to other unrelated cytotoxic agents.